A culture substratum appropriate for brain cells is a chondroitin sulfate glycosaminoglycan in serum.
When cells dissociated from the neonatal rat brains are plated on a poly-lysine-coated surface in a serum-free medium, they display a strange morphology: a dark and extended cell body. Preincubation of the surface with fetal bovine serum was found to inhibit the appearance of this strange contraction of the basal cell sheets in a dose-dependent manner. This finding indicated the presence of a factor(s) in the serum, which might be an appropriate substratum for prolonged survival of brain neurons. In the current study, this factor was highly purified through DEAE ion-exchange chromatography followed by gel filtration. The factor was eluted from a Superose column at fractions corresponding to a molecular weight greater than 1000 kDa. By SDS-PAGE analysis, these fractions were found to contain a major band (>/=1000 kDa) positive for alcian blue and few minor bands faintly stainable with Coomassie blue. The activity of the purified sample, inducing the morphological change in cells, was diminished by incubation with chondroitinase ABC. Neither heparitinase II, hyaluronidase, nor trypsin modified the activity. An authentic chondroitin sulfate (type B) mimicked the serum action on the morphology of brain cells in early stages of culture. Taking these findings together, it is suggested that the factor in serum beneficial for the attachment of brain cells is composed of a chondroitin sulfate with a Mr greater than 1000 kDa. Cortical cells dissociated from the neonatal rat brain attached well to the purified factor-coated surface and displayed a healthy morphology: an optically-reflective cell body with thick neurites for at least 3 days in the absence of serum.
Watanabe
M
M
Department of Molecular Physiology, Institute for Molecular and Cellular Regulation, Gunma University, Showa-Machi 3-39-15, Maebashi 371, Japan.
Ishikawa
K
K
Tatemoto
K
K
D000818:Animals; D000831:Animals, Newborn; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D002809:Chondroitin Sulfates / Q000097:blood* / Q000302:isolation & purification / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003470:Culture Media; D016895:Culture Media, Serum-Free; D004591:Electrophoresis, Polyacrylamide Gel; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D051381:Rats
D003470:Culture Media; D016895:Culture Media, Serum-Free; D002809:Chondroitin Sulfates